Cutaneous thermosensation is critical for detecting and avoiding potentially harmful environmental temperatures, as well as thermoregulation. This mechanism frequently goes awry under neuropathic pain conditions, manifesting as cold hypersensitivity, a condition in which normally cool input is perceived as painfully cold. A limited understanding of how cold stimuli are encoded in the nervous system has left this debilitating condition poorly managed. Recent studies suggest that cold input is conveyed from the periphery to the central nervous system by a population of primary afferents that express the temperature-sensitive cation channel TRPM8. However, the neural circuits through which this information is processed in the spinal cord and conveyed to the brain remain almost completely unknown. To address this fundamental gap in knowledge, this proposal will dissect the neural circuitry that encodes cold stimuli through a combination of behavioral, electrophysiological and morphological approaches. Based on our preliminary studies, we believe that cold input is conveyed from the spinal cord to the brain by a subset of neurokinin 1 receptor (NK1R)-expressing spinoparabrachial projection neurons. To investigate this possibility, our lab recently generated a NK1R-CreER allele, thereby providing us with genetic access to label and manipulate the activity of NK1R neurons. This proposal will test the hypothesis that cold input is conveyed from the spinal cord to the brain by a subset of morphologically distinct NK1R neurons that receive synaptic input (direct or indirect) from cold-sensing, TRPM8 primary sensory afferents. Aim 1 will investigate whether NK1R spinal projection neurons are required for aversion to cold using behavioral assays. Aim 2 will dissect the circuitry between TRPM8 primary afferents and NK1R spinal projection neurons that respond to cold stimuli using electrophysiology and optogenetic approaches in combination with our ex vivo somatosensory preparation. Aim 3 will determine the morphology of cold-selective spinal projection neurons by using histology to reconstruct cell architecture within the spinal cord dorsal horn. These experiments will establish the basic spinal circuitry that mediates cold input, thereby providing a framework from which to investigate changes in circuitry that underlie cold hypersensitivity in future studies. The success of this multifaceted fellowship training plan, which includes activities for scientific and career development, will be aided by the scientific expertise, collaboration, and educational opportunities offered by the Pittsburgh Center for Pain Research.